Polymerase chain reaction (PCR) protocols designed to rapidly and specifically detect Mycobacterium avium, M. intracellulare, and M. kansasii infections were developed. These protocols utilize PCR primers derived from unique sequences within the mycobacterial 16s rRNA genes. The M. avium, M. intracellulare, and M. kansasii PCR procedures specifically detect 1-10 fentograms of homologous genomic DNA. No PCR reactivity was demonstrated against DNA from 18 other strains of mycobacteria or from 7 closely related bacterial strains. Furthermore, bacterial titration experiments indicated that 1-10 nontuberculous mycobacterial bacilli could be directly detected using these PCR protocols. Currently, these PCR methodologies are being adapted for direct assays of clinical samples.